Binding assay method using a signal preventing reagent

Abstract

A device for performing an enzyme-labelled binding assay comprises an absorbent material and a developing solution containing a substrate for said enzyme, wherein the absorbent material is provided with a plurality of reagent zones including an indicator reagent zone and is capable of transporting the developing solution by capillary action sequentially through the reagent zones, and wherein the indicator reagent zone includes a reagent capable, directly or indirectly, of immobilizing an enzyme-labelled reagent in an amount dependent on the assay result, characterized in that the absorbent material includes a reagent that prevents a signal formation except where enzyme-labelled reagent is immobilized at the indicator reagent zone. The absorbent material is suitably in the form of an elongate strip provided with transverse reagent zones. The device is useful for performing immunoassays, including immunometric assays and dual analyte assays.

Claims

1. A method of performing an enzyme-labeled binding assay comprising:

(a) adjoining an absorbant material with a reservoir containing a developing solution comprising a substrate of an enzyme of an enzyme-labeled reagent that acts upon said substrate to produce a detectable signal, or to produce a substrate which in turn is acted upon by a signal-producing enzyme reaaent to produce a detectable signal,

said absorbent material being provided with a sample-receiving zone, a plurality of sequential and transverse reagent zones comprising immobilized or non-immobilized reagents, including an immobilized indicator reagent zone capable of directly or indirectly immobilizing said said enzyme-labeled reagent in an amount dependent on the assay result, and also including a reagent zone comprising an immobilized or non-immobilized reagent that prevents a signal formation except where the enzyme-labeled reagent is immobilized at the indicator reagent zone, wherein said reagent that prevents signal formation is in excess concentration relative to the concentration of the enzyme-labeled reagent and the substrate;

(b) applying a sample to be assayed to said sample receiving zone;

(c) applying the developing solution to said absorbing material so that as the developing solution advances through said absorbing material, the solvent front of said advancing developing solution proceeds sequentially through the reagent zones, and the non-immobilized reagents and the substrate co-migrate at the developing solution solvent front; and

(d) allowing the solvent front to proceed through and past the immobilized indicator reagent zone so that the reagent that prevents signal formation is removed from the immobilized indicator reagent zone and a signal is formed, wherein said signal is dependent on the assay result.

2. The method according to claim 1 wherein the absorbent material is in the form of an elongate strip with transverse reagent zones.

3. The method according to claim 2 wherein the absorbent material bibulous paper.

4. The method according to claim 1 wherein the reagent that prevents signal formation is (1) a reversible inhibitor of the enzyme-labeled reagent; (2) a further enzyme or a reagent which will compete with the enzyme-labeled reagent for substrate or cofactors; (3) an alternative substrate for the enzyme-labeled reagent which is not chromogenic and competes with the signal-producing substrate for the enzyme; (4) an end-product of a reaction catalyzed by the enzyme-labeled reagent which is able to inhibit the enzyme; or (5) a reagent which is able to maintain the pH in the locality of the enzyme-labeled reagent away from the pH optimum of the enzyme.

5. The method according to claim 4 wherein the reagent that prevents signal formation is an enzyme or reagent which will compete with the enzyme-labeled reagent for substrate or cofactors.

6. The method according to claim 4 wherein the reagent that prevents signal formation is a reductase or a reducing agent and the enzyme-labeled reagent is an oxidoreductase-labeled reagent.

7. The method according to claim 6 wherein the oxidoreductase is a peroxidase.

8. The method according to claim 7 wherein the peroxidase is horseradish peroxidase.

9. The method according to claim 4 wherein the reagent that prevents signal formation is catalase.

10. The method according to claim 4 wherein the reagent that prevents signal formation is ascorbic acid or a salt thereof.

11. The method according to claim 10 wherein the reagent that prevents signal formation is sodium ascorbate.

12. The method according to claim 1 for determining the relative concentrations of two sample analytes.

13. The method according to claim 12 for determining the relative concentrations of pregnanediol-3-glucuronide and oestrone-3-glucuronide in urine.

14. The method according to claim 1, which is a competitive binding assay.

15. The method according to claim 14 wherein a sample analyte competes with the enzyme labeled reagent for binding to the immobilized indicator reagent zone.

16. The method according to claim 14 wherein an analyte in the sample binds to an enzyme labeled reagent or binds to a reagent in competition with an enzyme labeled analyte analogue.

17. The method according to claim 1, which is a non-competitive binding assay.

18. The method according to claim 1 wherein the signal produced in the indicator reagent zone is fluorometric, chemiluminometric or calorimetric.